The field of this invention is screens for drugs effecting nuclear hormone receptor function.
Nuclear hormone receptors comprise a large, well-defined family of ligand-activated transcription factors which modify the expression of target genes by binding to specific cis-acting sequences (Laudet et al., 1992, EMBO J, Vol, 1003-1013; Lopes da Silva et al., 1995, TINS 18, 542-548; Mangelsdorfet al., 1995, Cell 83, 835-839; Mangelsdorf et al., 1995, Cell 83, 841-850). Family members include both orphan receptors and receptors for a wide variety of clinically significant ligands including steroids, vitamin D, thyroid hormones, retinoic acid, etc. Ligand binding is believed to induce a conformational change in the receptors and promote their association with transcriptional coactivators, which are a diverse group of large nuclear proteins (Glass et al., 1997, Curr Opn Cell Biol 9, 222-232), which may share a signature sequence motif (Heery et al., 1997, Nature 733-736). The resulting complex then binds high affinity sites in chromatin and modulates gene transcription.
The classic approach to identifying agonists or antagonists of nuclear hormone receptors is the ligand displacement assay, where the displacement of radiolabeled ligand by candidate agents is detected. An alternative approach is a cell-based transcription assay for expression of a reporter of nuclear hormone receptor activation (e.g. Evans et al. (1991) U.S. Pat. No. 5,071,773). More recently, a gel-based coactivator dependent receptor ligand assay (Krey et al., 1997, Mol Endocrinol 11, 779-791) has been used to identify ligands of peroxisome proliferator-activated receptors (PPARs), which are nuclear hormone receptors activated by a variety of compounds including hypolipidemic drugs. Unfortunately, these various assays suffer from a number of limitations including a required known ligand and time, labor and resource intensive cell-based and gel-based methods, respectively.
The invention provides methods and compositions for efficient screening of modulators of nuclear hormone receptor function, without the use of cell- or gel-based steps. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for bioactive compounds.
In one embodiment, the invention provides in vitro methods comprising the steps of (a) forming a mixture comprising a nuclear hormone receptor, a peptide sensor and a candidate agent, but not a natural coactivator protein of the receptor, wherein the sensor provides direct, in vitro binding to the receptor under assay conditions; (b) measuring an agent-biased binding of the sensor to the receptor; and (c) comparing the agent-biased binding with a corresponding unbiased binding of the sensor to the receptor, wherein a difference between the biased and unbiased bindings indicates that the agent modulates a receptor function. In particular embodiments, the sensor comprises an amphipathic alpha helix nuclear hormone interacting domain comprising a nuclear hormone transcriptional coactivator motif sequence. To ensure specificity and optimize binding, the sensor is generally present at sub-micromolar concentration and the binding reaction occurs in solution. In a preferred embodiment, the sensor comprises a fluorescent label and the measuring step comprises detecting fluorescence polarization of the label.
The invention also provides reagents such as labeled sensor peptides and reaction mixtures consisting essentially of nuclear hormone receptor, a peptide and a candidate agent, wherein the peptide provides direct, in vitro ligand-dependent binding to the receptor, especially in which the binding is enhanced in the presence of the agent.